Staphylococcus aureus (S. aureus) is a pathogenic bacterium that causes various diseases in humans and animals. Staphylococcus aureus contaminates foods, and upon proliferation produces exotoxins (enterotoxins). When foods containing enterotoxins are consumed, symptoms of acute gastroenteritis appear within 2-6 hours and are followed by vomiting, abdominal pain and diarrhea. In serious cases this is accompanied by lower grade fever, fall in marked symptoms of poisoning including blood pressure, intrathoracic anxiety, clouding of consciousness and pulse rate reduction, often requiring emergency hospital admission.
Methicillin-resistant Staphylococcus aureus (hereunder referred to as “MRSA”) is a type of Staphylococcus aureus causing hospital infections that have come to constitute a serious social problem. In recent years, various multiple drug-resistant MRSA species have appeared that exhibit resistance to many other antibiotics other than methicillin, including β-lactam agents such as penicillin-based and cephem-based antibiotics. Because MRSA has such multiple drug resistance, it is difficult to treat its infection. Staphylococcus aureus also includes methicillin-sensitive Staphylococcus aureus (hereunder referred to as “MSSA”).
While effective methods of treating MRSA have been developed in recent years, from the viewpoint of side-effects and preventing emergence of new resistant strains, it is considered undesirable to employ methods of treating MRSA-infected patients as standard treatment for patients infected with MSSA that does not have multiple drug resistance. That is, it is important to promptly identify MRSA and carry out appropriate measures for MRSA patients or MRSA-infected areas, and when Staphylococcus aureus has been detected, it is extremely important to reliably discern whether the strain is MRSA, or MSSA which does not have multiple drug resistance and requires different measures than MRSA, so that appropriate measures may be taken for each.
Conventional assessment of Staphylococcus aureus as MRSA or MSSA has been made by methods in which actual resistance to drugs is examined by culturing using a dilution method, disc sensitivity test or the like. However, such methods require long culturing times, and are associatcd with problems including variable results depending on various factors during culturing (inoculum concentration, culturing temperature, medium composition, drugs used, etc.), as well as the proficiency of the operating technician.
On the other hand, as means for discriminating MRSA/MSSA based on the presence of PBP2′, which is a new alternate enzyme for penicillin-binding proteins (PBP1, PBP2, PBP3 and PBP4), the characteristic proteins of MRSA, there have been proposed methods for detecting PBP2′ by nonradioactive testing (see Non-patent documents 1 and 2, for example) or radioimmunoassay and enzyme immunoassay methods using antibodies for PBP2′ (see Patent document 1 and Non-patent document 3, for example). However, such methods require the complex procedure of preparing an antigen-containing cell membrane fraction by ultracentrifugation, and they are difficult to carry out at ordinary examination facilities. In addition, such methods employ urea as a denaturing agent for extraction of the antigen, and therefore urea remains in the reaction system during subsequent immunoassay so that a measuring time of several hours is necessary, thus posing another inconvenience for routine examination.
There are also known methods for detection of mecA, the gene coding for PBF'2′ produced by multiple-drug-resistant Staphylococcus aureus, by PCR-based genetic engineering techniques, whereby MRSA is discriminated based on the presence of the gene in the test strain (see Non-patent document 4, for example). However, the presence of mecA does not necessarily reflect multiple drug resistance of Staphylococcus aureus, and Staphylococcus aureus strains are known to exist that do not acquire multiple drug resistance despite carrying the mecA gene.
On the other hand, methods are known for extracting PBP2′ antigen from MRSA without complex procedures such as ultracentrifugation of the cell membrane fraction, and such methods include extraction with alkali metal hydroxides, alkaline earth metal hydroxides or amine aqueous solutions (see Patent document 2, for example).